The background to the invention will be discussed in the context of the association of proteins. However, the scope of the invention should not be understood to be limited thereto.
Proteins do not act in isolation in a cell, but in stable or transitory complexes, with protein-protein interactions being key determinants of protein function (Auerbach et al., (2002), Proteomics, 2, 611-623). Furthermore, proteins and protein complexes interact with other cellular components like DNA, RNA and small molecules. Understanding both the individual proteins involved in these interactions and their interactions are important for a better understanding of biological processes.
Several tools exist for demonstrating protein-protein interactions either in vitro, coimmunoprecipitation with the potential for cross-linking at the cell surface, or in vivo, including for example the resonance energy transfer (RET) technologies of fluorescence RET (FRET) and bioluminescence RET (BRET).
The interaction of G-protein coupled receptors (GPCRs) represents an excellent example of the physiological and potential pharmacological relevance of the association of proteins, and in particular the relevance of hetero-dimers and/or -oligomers. As Milligan (Milligan, (2006), Drug Discovery Today, 11, 541-549) observes, homo-dimerisation and -oligomerisation have limited implications for the drug discovery industry, while “differential pharmacology, function and regulation of GCPR hetero-dimers and -oligomers suggest means to selectively target GPCRs in different tissues and hint that the mechanism of function of several pharmacological agents might be different in vivo than anticipated from simple ligand screening programmes that rely on heterologous expression of a single GPCR”.
Communoprecipitation has been used to identify GPCR heterodimers (Jordan B A & Devi L A (1999) G-protein-coupled receptor heterodimerization modulates receptor function Nature 399, 697-700). However, coimmunoprecipitation does not enable the distinction between constitutive and random associations. In particular, there is concern that artefactual aggregation occurs following cell lysis and solubilization (Kroeger K M et al. (2003) G-protein coupled receptor oligomerization in neuroendocrine pathways. Front. Neuroendocrinol. 24, 254-278). Further, coimmunoprecipitation is not amenable to automation or high throughput screening.
Fluorescence resonance energy transfer (FRET) is capable of detecting in vivo protein-protein interactions (Forster, (1948), Ann. Phys. 2, 57-75). This technique became particularly attractive and applicable to assays in living cells when the green fluorescent protein (GFP) and its mutant variants with different spectral characteristics were cloned. This allowed the genetic attachment of GFP and its variants to any target protein by fusing the encoding DNA sequences (Heim et al., (1994), PNAS. USA. 91, 12501-12504). FRET is able to monitor interactions that occur anywhere inside the cell. FRET can be determined in any cell type (mammalian, yeast, bacterial etc.) or cell-free system. It can be detected by fluorescence spectroscopy, fluorescence microscopy and fluorescence activated cell sorting (FACS).
Bioluminescence resonance energy transfer (BRET) is another technique that has been developed to study in vivo protein-protein interactions (Xu et al., (1999), PNAS. USA, 96, 151-156; Eidne et al., (2002), Trends Endocrin. Metabol. 13, 415-421). Like FRET, BRET allows detection within living cells or cell-free systems and is not restricted to a particular cellular compartment.
It is difficult to differentiate background signal from signals resulting from constitutive interactions using FRET or BRET to assess direct interactions between labelled proteins. Furthermore, interaction affinity does not relate directly to signal intensity as RET is dependent upon the relative orientation of and distance between the energy donor and acceptor.
‘Saturation’ BRET [Mercier J F, Salahpour A, Angers S, Breit A & Bouvier M (2002) Quantitative assessment of beta 1- and beta 2-adrenergic receptor homo- and heterodimerization by bioluminescence resonance energy transfer. J Biol Chem 277, 44925-44931.] has been suggested as a method to differentiate background and constitutive signals, however, this requires expression of increasing concentrations of acceptor-labelled protein relative to donor-labelled protein in order to generate saturation curves and is by no means high throughput.
The preceding discussion is intended only to facilitate an understanding of the invention. It should not be construed as in any way limiting the scope or application of the following description of the invention, nor should it be construed as an admission that any of the information discussed was within the common general knowledge of the person skilled in the appropriate art at the priority date.